Inhibitorily active recombinant human stefin B. Gene synthesis, expression and isolation of an inhibitory active MS-2 pol-stefin B fusion protein and preparation of Des[Met1,2(2)]stefin B

A synthetic gene coding for the human intracellular cysteine proteinase inhibitor, stefin B, was constructed from 13 chemically synthesized oligonucleotides according to the method of Khorana. The gene was inserted into the plasmid vector pTZ, amplified and sequenced. For expression, a temperature-inducible system producing fusion proteins was used. With the vector pEx31A containing the synthetic cystatin B gene, E. coli strain 537 produced a fusion protein of the N-terminal part of bacteriophage MS-2 polymerase and [Met-2Gly-1]stefin B. Lysates of the induced bacteria were inhibitorily active against papain. The fusion protein was expressed in high yield (about 20% of total E. coli proteins) and mostly deposited as inclusion bodies. The unfolded fusion protein was partially purified in the presence of urea. After refolding, approx. 6% of the protein was inhibitorily active against papain, human cathepsin H and B. Des[Met1,2(2)]stefin B was released by cyanogen bromide cleavage of the fusion protein and identified by N-terminal amino-acid sequence analysis. The non-separated cleavage products were also inhibitorily active after refolding. The estimated inhibition constants for the fusion protein and its cleavage products were similar to those reported for natural stefin B.     

Differences in the effects of TFE on the folding pathways of human stefins A and B.

Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.     

Interaction of human stefin B in the prefibrillar oligomeric form with membranes. Correlation with cellular toxicity

Protein aggregation is central to most neurodegenerative diseases, as shown by familial case studies and by animal models. A modified 'amyloid cascade' hypothesis for Alzheimer's disease states that prefibrillar oligomers, also called amyloid-beta-derived diffusible ligands or globular oligomers, are the responsible toxic agent. It has been proposed that these oligomeric species, as shown for amyloid-beta, beta2-microglobulin or prion fragments, exert toxicity by forming pores in membranes, initiating a cascade of detrimental events for the cell. Interaction of granular aggregates and globular oligomers of an amyloidogenic protein, human stefin B, with model lipid membranes and monolayers was studied. Prefibrillar oligomers/aggregates of stefin B are shown to cause concentration-dependent membrane leaking, in contrast to the homologous stefin A. Prefibrillar oligomers/aggregates of stefin B also increase the surface pressure at an air-water interface, i.e. they have amphipathic character and are surface seeking. In addition, they show stronger interaction with 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] monolayers than native stefin A or nonaggregated stefin B. Prefibrillar aggregates interact predominantly with acidic phospholipids, such as dioleoylphosphatidylglycerol or dipalmitoylphosphatidylserine, as shown by calcein release experiments and surface plasmon resonance. The same preparations are toxic to neuroblastoma cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, again in contrast to the homologue stefin A, which does not aggregate under any of the conditions studied. This study is aimed to contribute to the general model of cellular toxicity induced by prefibrillar oligomers of amyloidogenic proteins, not necessarily involved in pathology.     

The cross-road between the mechanisms of protein folding and aggregation; study of human stefin B and its H75W mutant

The role of the aromatic residue at site 75 to protein stability, the mechanism of folding and the mechanism of amyloid-fibril formation were investigated for the human stefin B variant (bearing Y at site 31) and its point mutation H75W. With an aim to reveal the conformation at the cross-road between folding and aggregation, first, the kinetics of folding and oligomer formation by human stefin B(Y31) variant were studied. It was found to fold in three kinetic phases at pH 4.8 and 10% TFE; the pH and solvent conditions that transform the protein into amyloid fibrils at longer times. The same pH leads to the formation of native-like intermediate (known from previous studies of this variant), meaning that the process of folding and amyloid-fibril formation share the same structural intermediate, which is in this case native-like and dimeric. At pH 5.8 and 7.0 stefin B folded to the native state in four kinetic phases over two intermediates. In distinction, the mutant H75W did not fold to completion, ending in intermediate states at all pH values studied: 4.8, 5.8 and 7.0. At pH 4.8 and 5.8, the mutant folded in one kinetic phase to the intermediate of the “molten globule” type, which leads to the conclusion that its mechanism of folding differs from the one of the parent stefin B at the same pH. At pH 7.0 the mutant H75W folded in three kinetic phases to a native-like intermediate, analogous to folding of stefin B at pH 4.8.     

Amyloid fibril formation by human stefin B in vitro: immunogold labelling and comparison to stefin A

The mechanism by which proteins form amyloid fibrils is of high interest to the scientific community as its understanding could resolve questions relevant to conformational diseases. The structural and energetic basis of the process is still largely unknown. The main controversial issue is the co-existence of several protein conformations. Three models for the mechanism of protein fibrillogenesis have been proposed which need to be tested by experiments. In this report, amyloid fibrils grown from human stefin B (type I cystatin) are described. This physiologically relevant protein readily forms fibrils in vitro, in contrast to the homologue--human stefin A--which forms fibrils under extreme conditions only. In order to specifically label stefin B fibrils in vitro, rabbit polyclonal antibody and mouse monoclonal antibody A6/2 against human stefin B were used for immunogold labelling. Samples were examined by transmission electron microscopy. Fibrils of stefin B were strongly labelled using polyclonal antibody and Protein A gold, whereas no positive reaction was observed with monoclonal antibody A6/2.     

Differences in aggregation properties of three site-specific mutants of recombinant human stefin B

We describe expression, purification, and characterization of three site-specific mutants of recombinant human stefin B: H75W, P36G, and P79S. The far- and near-UV CD spectra have shown that they have similar secondary and tertiary structures to the parent protein. The elution on gel-filtration suggests that recombinant human stefin B and the P36G variant are predominantly monomers, whereas the P79S variant is a dimer. ANS dye binding, reflecting exposed hydrophobic patches, is highest for the P36G variant, both at pH 5 and 3. ANS dye binding also is increased for stefin B and the other two variants at pH 3. Under the chosen conditions the highest tendency to form amyloid fibrils has been shown for the recombinant human stefin B. The P79S variant demonstrates a longer lag phase and a lower rate of fibril formation, while the P36G variant is most prone to amorphous aggregation. This was demonstrated by ThT fluorescence as a function of time and by transmission electron microscopy.     

Chemical synthesis of a gene for human stefin A and its expression in E. coli

A DNA containing the coding sequence for the human cysteine proteinase inhibitor stefin A was obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method. The 306-bp synthetic gene carries signals for the initiation and termination of its translation. The gene was expressed in E. coli using a cytoplasmic expression vector and stefin A was secreted under the control of the E. coli alkaline phosphatase signal sequence, respectively. The secreted hybrid protein was shown to exhibit biological properties similar to the native protein isolated from human plasma.     

High affinity copper binding by stefin B (cystatin B) and its role in the inhibition of amyloid fibrillation

We show that human stefin B, a protease inhibitor from the family of cystatins, is a copper binding protein, unlike stefin A. We have used isothermal titration calorimetry to directly monitor the binding event at pH 7 and pH 5. At pH 7 stefin B shows a picomolar affinity for copper but at pH 5 the affinity is in the nanomolar range. There is no difference in the affinity of copper between the wildtype stefin B (E31 isoform) and a variant (Y31 isoform), whereas the mutant (P79S), which is tetrameric, does not bind copper. The conformation of stefin B remains unaltered by copper binding. It is known that below pH 5 stefin B undergoes a conformational change and amyloid fibril formation. We show that copper binding inhibits the amyloid fibril formation and, to a lesser degree, the initial aggregation. Similarities to and differences from other copper binding amyloidogenic proteins are discussed.     

Different propensity to form amyloid fibrils by two homologous proteins-Human stefins A and B: searching for an explanation

By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 3 nm and longitudinal repeat of 25-27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik et al., Biochem Biophys Acta 2002;1594:1-5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.     

Interaction between Oligomers of Stefin B and Amyloid-�� in Vitro and in Cells

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-β-(1–40) peptide (Aβ). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Aβ is oligomer specific. The dimers and tetramers of stefin B, which bind Aβ, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Aβ fibril formation. When expressed in cultured cells, stefin B co-localizes with Aβ intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Aβ epitope. Thus, stefin B is another APP/Aβ-binding protein in vitro and likely in cells.     

Major differences in stability and dimerization properties of two chimeric mutants of human stefins

Stefins A and B are cysteine proteinase inhibitors that have considerable sequence similarity but marked differences in their stability and folding properties. Two chimeric proteins were designed to shed light on these differences. The chimeric mutants have been expressed in Escherichia coli and have been isolated. The first, A37B, consists of 37 residues of stefin A, comprising the N-terminal and the alpha-helix, joined to 61 residues of stefin B; the second, A61B, consists of 61 N-terminal residues of stefin A, followed by 37 residues of stefin B. Spectroscopic properties of the chimeric proteins (absorption, CD, and NMR spectra), together with activity measurements, have confirmed that both have well-defined tertiary structure and are active as cysteine proteinase inhibitors. Characterization consisted of GuHCl denaturation, ANS binding as a function of pH, and monitoring of dimerization under partially denaturing conditions. The c(m) values are 1.3 M GuHCl for A61B as compared with 2.7 M GuHCl for stefin A, and 2.1 M GuHCl for A37B as compared with 1.4 M GuHCl for stefin B (all at pH 7.5, 25 degrees C). However (G degrees (N-U) is lower for both chimeric proteins (18 +/- 3 kJ/mol) than for the parent stefins (28 +/- 3 kJ/mol). In pH denaturation, unlike stefin B, neither chimeric mutant unfolds to I(N) below pH 5.4. At pH 3, where stefin B forms a molten globule and stefin A is native, both A37B and A61B show increased ANS fluorescence and aggregate visibly. Dimers at pre-denaturation conditions are observed in all the proteins under study, but they remain "trapped" only in stefin A.     

Interaction with model membranes and pore formation by human stefin B: studying the native and prefibrillar states

Human stefin B, from the family of cystatins, is used as a model amyloidogenic protein in studies of the mechanism of amyloid fibril formation and related cytotoxicity. Interaction of the protein's prefibrillar oligomers/aggregates with predominantly acidic phospholipid membranes is known to correlate with cellular toxicity. In the present study, we measured membrane interaction of the prefibrillar and native states for three variants: the Y31 isoform studied previously, the wild-type protein and the G4R mutant; the latter is observed in progressive myoclonus epilepsy of type 1. In addition to using critical pressure and surface plasmon resonance, we assessed membrane permeabilization by calcein release and electrophysiological measurements. It was demonstrated for the first time that wild-type stefin B and the Y31 isoform are able to form pores in planar lipid bilayers, whereas G4R destroys the bilayer by a non pore-forming process. Similarities to other amyloidogenic proteins and the possible physiological implications of our findings are discussed.     

Functional expression of recombinant human stefin A in mammalian and bacterial cells

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-1 monkey kidney cells utilizing pET and recombinant vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and Western blot analysis utilizing a polyclonal antibody against rat cystatin alpha. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A ( approximately 10kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BS-C-1 cells, was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells, on the other hand, can provide a significant research tool to study the functional roles of stefin A in mammalian systems such as regulation of cysteine proteases.     

Gain in toxic function of stefin B EPM1 mutants aggregates: Correlation between cell death,aggregate number/size and oxidative stress

EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death.We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.     

Inhibitory properties of low molecular mass cysteine proteinase inhibitors from human sarcoma

Elevated activities of cysteine proteinases such as cathepsins B and L and cancer procoagulant have been linked to tumor malignancy. In the present study we examined the hypothesis that these elevated activities could be due to impaired regulation by the endogenous low molecular mass cysteine proteinase inhibitors (cystatins). Inhibitors from human sarcoma were compared to those from human liver, a normal tissue in which the inhibitors had been characterized previously. An extract of cystatins from sarcoma was less effective against papain and cathepsin B (liver or tumor) than was an extract from liver. This reduced inhibitory capacity in sarcoma was not due to a reduction in either the concentrations or specific activities of the cystatins or an absence of any family or isoform of cystatins. We purified two members of the cystatin superfamily (stefin A and stefin B) to homogeneity and determined their individual inhibitory properties. Stefins B from liver and sarcoma exhibited comparable inhibition of papain and cathepsin B. In contrast, stefin A from sarcoma exhibited a reduced ability to inhibit papain, human liver cathepsins B, H and L and human and murine tumor cathepsin B. The Ki for inhibition of liver cathepsin B by sarcoma stefin A was 10-fold higher than that for inhibition of liver cathepsin B by liver stefin A, reflecting a reduction in the rate constant for association and an increase in the rate constant for dissociation. Cancer is now the third pathologic condition reported to be associated with alterations in cystatins, the other two being amyloidosis and muscular dystrophy.     

Human Stefin B Role in Cell's Response to Misfolded Proteins and Autophagy

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.     

Direct evidence for excitonically coupled chlorophylls a and b in LHC II of higher plants by nonlinear polarization spectroscopy in the frequency domain

Human stefin B (cystatin B) is an intracellular cysteine proteinase inhibitor broadly distributed in different tissues. Here, we show that recombinant human stefin B readily forms amyloid fibrils in vitro. It dimerises and further oligomerises, starting from the native-like acid intermediate, I(N), populated at pH 5. On standing at room temperature it produces regular (over 4 microm long) fibrils over a period of several months. These have been visualised by transmission electron microscopy and atomic force microscopy. Their cross-sectional diameter is about 14 nm and blocks of 27 nm repeat longitudinally. The fibrils are smooth, of unbranched surface, consistent with findings of other amyloid fibrils. Thioflavin T fluorescence spectra as a function of time were recorded and Congo red dye binding to the fibrils was demonstrated. Adding 10% (v/v) trifluoroethanol resulted in an increased rate of fibrillation with a typical lag phase. The finding that human stefin B, in contrast to the homologue stefin A, forms amyloid fibrils rather easily should promote further studies of the protein's behaviour in vivo, and/or as a model system for fibrillogenesis.     

On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60 ± 5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved “burst” phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25 s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6 s) detected by all the spectroscopic probes, which occurred subsequent to an initial “burst” phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed. Protein 32:296–303, 1998. © 1998 Wiley-Liss, Inc.     

Putative alternative functions of human stefin B (cystatin B): binding to amyloid‐beta,membranes, and copper

We describe studies performed thus far on stefin B from the family of cystatins as a model protein for folding and amyloid fibril formation studies. We also briefly mention our studies on aggregation of some of the missense EPM1 mutants of stefin B in cells, which mimic additional pathological traits (gain in toxic function) in selected patients with EPM1 disease. We collected data on the reported interactors of stefin B and discuss several hypotheses of possible cytosolic alternative functions.     

Essential role of proline isomerization in stefin B tetramer formation

Here we present the tetrameric structure of stefin B, which is the result of a process by which two domain-swapped dimers of stefin B are transformed into tetramers. The transformation involves a previously unidentified process of extensive intermolecular contacts, termed hand shaking, which occurs concurrently with trans to cis isomerization of proline 74. This proline residue is widely conserved throughout the cystatin superfamily, a member of which, human cystatin C, is the key protein in cerebral amyloid angiopathy. These results are consistent with the hypothesis that isomerization of proline residues can play a decisive role in amyloidogenesis.